|
| Status |
Public on Dec 18, 2008 |
| Title |
XRLP (xrn1 rrp6 lsm1 pat1) Mutant Salt Shock 30min rep1 (Watson strand; Total RNA) |
| Sample type |
RNA |
| |
|
| Source name |
S.cerevisiae experimental RNA
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
Strain Background is FY23 x FY86 (Winston et al. 1995), carrying the auxotrophic markers: his3-delta200, leu2-delta1, trp1-delta63, ura3-52. The XRLP mutant contains deletions in the genes XRN1, RRP6, LSM1, and PAT1.
|
| Growth protocol |
Our unpublished studies suggested that among two dozen or so different conditions that we assayed, exposure to high salt (0.8M NaCl) results in the expression of the greatest fraction of known and novel transcripts, and thus was chosen as the experimental condition to use to find previously unannotated and low abundance transcripts. Cells were grown at 30 degrees C in YPD to approximately 1x10^7 cells/ml as determined by a Beckman Coulter Z2 Particle Count and Size Analyzer. 1.6M NaCl (in YPD) was added in an equal volume of YPD prewarmed to 30 degrees C (final concentration 0.8M). Cells were harvested after 30 minutes by filtration, frozen in liquid nitrogen, and kept at minus 80 degrees C until RNA extraction and purification.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from the cells using a slightly modified version of the traditional hot phenol protocol (Schmitt et al. 1990) followed by ethanol precipitation and washing. Briefly, 5ml of lysis buffer (10mM EDTA ph 8.0, 0.5% SDS, 10mM Tris-HCl pH 7.5) and 5ml of acid phenol were added to frozen cells and incubated at 60 degrees C for 1 hour with occasional vortexing, then placed on ice. The aqueous phase was extracted after centrifugation and additional phenol extraction steps were performed as needed, followed by a chloroform extraction. Total RNA was precipitated from the final aqueous solution with 10% volume 3M sodium acetate pH 5.2 and ethanol, and resuspended in nuclease-free water.
|
| Label |
biotin
|
| Label protocol |
Total RNA samples were prepared following the protocol exactly as described in David et al. PNAS 103(14):5320-5 (2006). PolyA RNA samples were prepared as follows. 500ug of total RNA were polyA purified using Qiagen Oligotex suspension to produce approximately 10ug of polyA RNA as determined by OD260/280. 2ug of the polyA purified RNA were then used in the generation of cDNA as per Affymetrix First Strand and Second Strand Synthesis protocols utilizing a T7-Oligo(dT) as the primer for the First Strand, followed by in vitro transcription to generate biotin labeled cRNA, as outlined by Affymetrix protocols. The cRNA was fragmented as described by Affymetrix, and then sent for hybridization and scanning by the PAN facility at Stanford University.
|
| |
|
| Hybridization protocol |
Hybridization was handled by the PAN facility at Stanford University according to Affymetrix protocols.
|
| Scan protocol |
Scanning was handled by the PAN facility at Stanford University according to Affymetrix protocols.
|
| Description |
XRLP (xrn1 rrp6 lsm1 pat1) Mutant Salt Shock 30min rep1 (Watson strand; Total RNA)
|
| Data processing |
Output files were generated post hybridization and scanning by Affymetrix software and the resulting files can be viewed using the Affymetrix Genome Browser software.
|
| |
|
| Submission date |
Jun 16, 2008 |
| Last update date |
Dec 18, 2008 |
| Contact name |
Gavin Sherlock |
| E-mail(s) |
sherlock@genome.stanford.edu
|
| Phone |
650 498 6012
|
| Fax |
650 724 3701
|
| URL |
http://genetics.stanford.edu/~sherlock/
|
| Organization name |
Stanford University
|
| Department |
Genetics
|
| Street address |
300 Pasteur Drive
|
| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305-5120 |
| Country |
USA |
| |
|
| Platform ID |
GPL7250 |
| Series (2) |
| GSE11800 |
Annotating Low Abundance and Transient RNAs in Yeast using Tiling Microarrays |
| GSE11802 |
Annotating Low Abundance and Transient RNAs in Yeast using Tiling Microarrays and Ultra High-throughput Sequencing |
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